Opioid Antagonists from the Orvinol Series as Potential Reversal Agents for Opioid Overdose

The opioid crisis continues to claim many lives, with a particular issue being the ready availability and use (whether intentional or accidental) of fentanyl and fentanyl analogues. Fentanyl is both potent and longer-acting than naloxone, the standard of care for overdose reversal, making it especially deadly. Consequently, there is interest in opioid reversal agents that are better able to counter its effects. The orvinol series of ligands are known for their high-affinity binding to opioid receptors and often extended duration of action; generally, compounds on this scaffold show agonist activity at the kappa and the mu-opioid receptor. Diprenorphine is an unusual member of this series being an antagonist at mu and only a partial agonist at kappa-opioid receptors. In this study, an orvinol antagonist, 14, was designed and synthesized that shows no agonist activity in vitro and is at least as good as naloxone at reversing the effects of mu-opioid receptor agonists in vivo.


■ INTRODUCTION
The ongoing worldwide opioid epidemic has been exacerbated by the arrival of fentanyl (and fentanyl analogues) on the illicit market. Fentanyl is a potent mu-opioid receptor (MOR) agonist and significantly contributes to the death toll resulting from the use of opioids. 1 Fentanyl is more resistant to reversal than many standard opioids 2 and may require multiple doses of naloxone after overdose. 3,4 For this reason, a higher dose formulation has recently been approved by the FDA (https:// www.fda.gov/news-events/press-announcements/fdaapproves-higher-dosage-naloxone-nasal-spray-treat-opioidoverdose).
The orvinols are a series of compounds targeting opioid receptors, initially synthesized as analgesics without the undesirable side effects associated with morphine and other typical opioid analgesics. 5 Generally, orvinols bind equally well to each of the three classical opioid receptors, MOR, kappa (KOR), and delta (DOR), with analogues showing different abilities to activate each opioid receptor type: for example, buprenorphine (1a) (MOR partial agonist and KOR/DOR antagonist), etorphine, and dihydroetorphine (2, 1b) (full agonists at MOR, KOR, and DOR) and diprenorphine (1c) (MOR antagonist and DOR/KOR partial agonist). 6 In veterinary practice, diprenorphine is a reversal agent (Revivon) for etorphine-induced immobilization, and 11 C-diprenorphine is utilized as a positron emission tomography imaging ligand for labeling opioid receptors in both preclinical and human clinical studies. Diprenorphine could be used as an improved overdose reversal agent due to its quick onset and its effectiveness in reversing fentanyl-and morphine-induced respiratory depression. 2 However, while often described as a MOR, KOR, and DOR opioid antagonist, diprenorphine shows partial agonist activity at both KOR and DOR; 6 the former is substantial enough to cause psychotomimetic effects in humans, 7 limiting its utility. Therefore, an improved rescue medication should retain diprenorphine's MOR antagonism, rapid onset, and potency versus fentanyl but with reduced KOR agonist activity. To that end, we synthesized and evaluated C7β-methyl analogues of diprenorphine as it has previously been found that adding a C7β-methyl group to the orvinols reduces KOR efficacy without significantly affecting MOR activity. 8 ■ RESULTS AND DISCUSSION Chemistry. We have previously reported on the synthesis of C7β-methyl analogues of the orvinols using a Lewis acidcatalyzed reaction between N-cyclopropylcarbonylnorthebaine and methacrolein. 8 There were two key issues with this approach, the first being that the scope was limited to Nacylated analogues of thebaine and the second being that while the overall yield of the Diels−Alder step was high, the desired 7β-methyl isomer was obtained only in a ratio of 2:3 with the undesired 7α-methyl epimer. While we and others 8,9 have determined that thebaine (3) reacts poorly with methacrolein under standard conditions (organic solvent and heat), Maat et al. 10 had reported the successful reaction of thebaine with a closely related dienophile, methyl methacrylate, simply using an extended reaction time (2 weeks) and heat (100°C). In our hands, attempting this method led to extensive polymerization and very low yields, but it did, in the case of methacrolein, provide some products with a favorable ratio of diastereomeric products. As the use of aqueous systems to promote Diels− Alder reactions is well known, 11 there seemed a reasonable possibility of improving these low yields. We have now found that there is a powerful aqueous solvent effect for this particular Diels−Alder reaction and that good yields (93% overall) and a far more favorable ratio of isomers can be obtained by carrying out the reaction in saturated brine at 80°C (66% of C7β-Me 4b/33% C7α-Me 4a as determined by the ratio of aldehyde protons in 1 H NMR). Thus, a much higher yield of the desired epimer is achieved, and by retaining the N−Me group, this method allows for manipulation of this Nsubstituent by standard methods later in the synthesis. Importantly, the reaction with methacrolein has proven to be reproducible in our hand, although not so with other dienophiles.
After isolation of the desired C7β-Me isomer 4b, the addition of MeLi gave a mixture of diastereomeric 2°alcohols (5, in a 2:1 ratio by NMR) that were hydrogenated over Pd/C to reduce the bridge (6) and then oxidized to methyl ketone (7). Further treatment with MeLi and subsequent 3-Odemethylation furnished the C7β-Me tertiary alcohol (9) (Scheme 1).
To access the N-cyclopropylmethyl series (Scheme 2), Ndemethylation of the Diels−Alder adduct (4b) followed by alkylation with cyclopropylmethyl bromide gave the key intermediate (11). Reduction afforded the 1°alcohol (12) with subsequent hydrogenation of the bridge and 3-Odemethylation, yielding 14. Alternatively, treatment of 11 with MeLi gave diastereomeric 2°alcohols 15 (in a 3:1 ratio by 1 H NMR), subsequently hydrogenated to 16a and 16b, which could be separated by column chromatography and then converted into their phenolic counterparts 17a and 17b. Otherwise, the diastereomeric mixture of 16a and 16b could be O-demethylated with the products 17a and 17b and then separated by silica gel chromatography. The stereochemistry of the secondary alcohols was assigned based on the crystal structure of the individual C20-diastereoisomer 16a (Supporting Information). Oxidation of 2°alcohols (16) (Table 1) with minimal selectivity. In fact, compounds 14, 17a, and 17b, where R 1 = cyclopropylmethyl, showed subnanomolar affinity at all three opioid receptors. The N−Me analogue (9) maintained the lack of selectivity but with a somewhat lower affinity for all three receptors. This lack of selectivity is consistent with the previous reports on the C7β-Me series 8 and with standard orvinols. 5,12,13 The relative agonist activity of the compounds was determined using the [ 35 S]GTPγS assay in the same membrane homogenates as employed for the binding assays. Table 2 shows the potency of the compounds as EC 50 values and the maximal effect of each compound compared to standard agonists DAMGO (MOR), U69593 (KOR), and SNC80 (DOR). As expected from previous reports, diprenorphine did not stimulate [ 35 S]GTPγS binding to cell membrane homogenates expressing MOR but showed partial agonist activity at KOR and DOR. Analogue 9, which has a methyl substituent on the tertiary N atom, was a potent partial agonist at MOR (Table 2). At KOR, 9 showed no agonist activity but was a low-potency partial agonist at DOR.
Compound 20 in which the N−Me group of 9 is replaced with N-cyclopropylmethyl retained partial agonist activity at MOR and DOR, with lower potency at MOR but improved potency at DOR. Like 9, compound 20 did not activate KOR. In the previously reported C20-phenyl secondary alcohol series having a C7β-Me substituent, 8 the most significant piece of structure−activity relationship (SAR) was the reduction in efficacy at KOR compared to C7β-H counterparts. Therefore, we did predict that 20 [the C7β-Me analogue of diprenorphine (1c)] would show lower KOR efficacy than the parent diprenorphine (1c). 20 is, in fact, a low efficacy partial agonist at MOR and DOR but a KOR antagonist, whereas diprenorphine (1c) is a DOR and KOR partial agonist and a MOR antagonist.
Knowing that the addition of any steric bulk to C20 would increase efficacy, particularly at MOR and KOR, 12 we investigated the effect of moving between 1, 2, and 3°methyl alcohols at this position. 17a and 17b, the diastereomeric secondary alcohols, were partial agonists at MOR and KOR, with little-to-no observable agonist stimulation at DOR in the [ 35 S]GTPγS assay. The corresponding secondary alcohol orvinols with C7β-H (21a and 21b) and the primary alcohol 21c have previously been described as potent antagonists with minimal efficacy at MOR, KOR, or DOR based on their lack of antinociceptive efficacy in the rat tail pressure and abdominal stretch assays. 14 The only compound in the current 7β-Me series that showed no ability to stimulate [ 35 S]GTPγS binding at MOR, KOR, or DOR was the 1°alcohol 14. Yet, 14 bound with high affinity to each receptor, including an 8-fold higher affinity at MOR than naloxone, indicating nonspecific antagonist activity at the opioid receptors. The fact that 14 and naloxone are both MOR antagonists means that their affinities measured by ligand binding assay will match affinities measured by pharmacological assay, indicating that 14 is a more effective antagonist. We therefore examined the ability of 14 and naloxone to reverse MOR-mediated antinociception and respiratory depression in mice. For MOR-mediated antinociception, we chose to use the highly potent, long-lasting agonist BU72, which we have previously shown in the mouse warm-water tail-withdrawal (WWTW) assay is fully effective at a dose of 0.32 mg/kg and exhibits an antinociceptive action for at least 5 h 15 (Figure 1), thus allowing us to readily differentiate the time course of naloxone and 14. When given at the peak effect of BU72 (1 h), both naloxone and 14 (1 mg/kg) fully reversed the antinociceptive effects of BU72 ( Figure 1). The antinociceptive effects completely returned 2 h after naloxone administration. In contrast, 14 continued to partially antagonize BU72 for as long as its antinociceptive action was evident. The success of 14 in reversing the antinociceptive effects of BU72 prompted us to evaluate its ability to reverse fentanyl (10 mg/kg, i.p.)-induced respiratory depression as measured by blood oxygen levels ( Figure 2). Again, we compared 14 with the FDA-approved standard of care, naloxone; other compounds, in particular, diprenorphine are more potent than naloxone, but diprenorphine is not approved for opioid overdose reversal in humans and has kappa-and delta-opioid agonist activity which could confound our findings. We used fentanyl as the orthosteric agonist in this case due to its strong respiratory depressant action that contributes significantly to opioid overdose deaths. We found that 14 (1 mg/kg i.p.) reversed the fentanyl-induced decrease in oxygen saturation in a manner comparable to the same dose of naloxone ( Figure 2). Both compounds had rapid onset. There was no significant difference in the effectiveness of the two compounds. However, since 14 was longer lasting than naloxone in the WWTW assay, it is possible that complete dose−response curves using lower doses of the antagonists might uncover differences. This is relevant since doses used in mice will not necessarily translate to humans where much lower doses are effective. For example, the dose of naloxone used to reverse opioid overdose in humans is 4−8 mg intranasally. 16 In this report, we expand upon SAR around the C7β-Me orvinol series and identify 14 as a high-affinity pan opioid antagonist that reverses MOR-mediated antinociception and respiratory depression. Considering both the results reported here and those reported previously for 22 and analogues, 8 the C7β-Me series as a whole has similar efficacy to the C7β-H series at MOR. Interestingly, the N−Me containing (9) had only ∼1.5 times higher efficacy at MOR than the N-CPM containing 17a, 17b, and 20. Therefore, the change in efficacy between N−Me and N-CPM in this series appears to be more modest than found in the closely related 7,7-spiro series 17 or in the standard orvinol 5,14 or the morphinan series (e.g., compare oxymorphone with naltrexone). At KOR, the 7β-Me series overall has lower efficacy than standard orvinols (C7β-H) but similar SAR, in which efficacy is higher for 2°alcohols than for 3°alcohols; Greedy et al. 12 showed that 2°alcohols in the standard series have higher efficacy than 3°alcohols. At DOR, the emerging evidence is that the 7β-Me ligands have lower efficacy than the standard orvinols.

■ CONCLUSIONS
The new Diels−Alder conditions reported in this paper allow access to a greater range of N-substituents in the C7β-Me series due to easy access to the nor-intermediate 10.
The new conditions also improve the β-Me/α-Me ratio, with a resultant increase in the yield of the desired isomer. Of the compounds synthesized and evaluated, 14 stands out as a potent antagonist across all three receptors, which is unique among the orvinol series. In the mouse WWTW assay, 14 (1 mg/kg) was able to reverse the effects of the long-acting MOR agonist BU72 more effectively and for longer than the same dose of the FDAapproved reversal agent naloxone and was able to reverse fentanyl-induced respiratory depression. The improved profile of 14 over naloxone is likely due, at least in part, to its higher affinity for MOR. Whether the complex pharmacokinetics of the orvinol series that can result in long half-lives plays a role is an avenue for future investigation. Overall, our data suggests that 14 could be the starting point for the development of a novel rescue therapy. ■ METHODS Chemistry. Synthesis. All reactions were carried out under nitrogen unless otherwise stated. Reagents and solvents were purchased from Sigma-Aldrich or Alfa Aesar and used without further purification. 1 H and 13 C NMR spectra were obtained using a Bruker 400 MHz instrument ( 1 H at 400 MHz, 13 C at 100 MHz); δ is given in ppm, J, in Hz. Column chromatography was performed using RediSep prepacked columns with a Teledyne Isco CombiFlash instrument. Ligands were tested as their hydrochloride salts, prepared by adding 2.5 equiv of HCl (1 N solution in diethyl ether) to a solution of the compound in anhydrous diethyl ether.
HPLC−ESI−TOF Analysis. HPLC−ESI−TOF analysis was conducted using an electrospray time-of-flight (MicrOTOF) mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), which was coupled to an Agilent HPLC stack (Agilent, Santa Clara, CA, United States) consisting of an Agilent G1312A binary pump with a G1329A autosampler and G1316A column oven. Analyses were performed in ESI positive and negative modes. The capillary voltage was set to 4500 V, the nebulizing gas at 2.2 bar, and the drying gas at 10.2 L/min at 220°C in each case. The TOF scan range was from 50 to 700 mass-to-charge ratio (m/z). The system was configured with a switching valve to perform flow injections or chromatography. In each case, 10 μL injections were made. The TOF was calibrated with a 10 μL sodium formate calibrant solution injection prior to the chromatographic/flow injection run. The calibrant solution consisted  of three parts of 1 M NaOH to 97 parts of 50:50 water/isopropanol with 2% formic acid. Automated data processing was performed using the Compass Data Analysis software scripts (Bruker Daltonik GmbH, Bremen, Germany).